The long-term objective of this proposal is to characterize and target those molecular events responsible for one of the most common, profound, and intriguing phenotypes of malignant tissues, i.e., their elevated capacity to utilize glucose, a critical carbon and energy source essential for cell growth. In earlier studies we identified hexokinase, the initial enzyme of glucose catabolism, as a major player and showed that, of its four major isozymes,only Type II is both highly overexpressed in tumors and bound to the mitochondria. Here, the enzyme is unregulated and produces the key metabolic precursor Glu-6-P at high rates. More recently while working at the gene level, we have made some very novel, definitive, and exciting progress, which for the first time in more than 6 decades, begins to shed light on the underlying cause of the highly glycolytic phenotype characteristic of numerous human cancers. Thus, we have shown that the Type II hexokinase gene is amplified, and upon isolating and characterizing its promoter in detail, demonstrated that it is activated, not only by glucose, insulin, glucagon, and cAMP, but also by both hypoxic conditions (common within highly malignant tumors), and by a mutant form of the tumor suppressor p53. These preliminary studies form a firm foundation for the studies proposed here which are focused on better understanding how the cancer- related Type II hexokinase gene is regulated, and how it can be successfully inhibited. Specific aims are 4-fold and will be to: 1) Establish how the Type Il hexokinase gene, silent in many normal cells, e.g., hepatocytes, is "switched on" during tumorigenesis; 2) Elucidate the molecular basis underlying the activation of the Type II hexokinase gene promoter by glucose; 3) Gain greater insight into the molecular basis and significance of the novel finding, that within tumor cells, the promoter for Type II hexokinase is activated by a mutant form of p53; and 4) Identify methods for arresting the growth of highly malignant cancers by selectively inhibiting the expression of the Type II hexokinase gene using antisense RNA and targeted gene disruption techniques. These studies are fundamental to our understanding at the gene level of one of the most common phenotypes of cancer cells, and are likely to lead to novel approaches for controlling the growth of highly malignant tumors.